Destabilising domains for conditionally stabilising a protein

ABSTRACT

The present disclosure relates to mutant polypeptides derived from Escherichia coli dihydrofolate reductase (DHFR) which can be fused to a polypeptide of interest for efficient conditional modulation of its activity. Also disclosed are polynucleotides encoding such mutant polypeptides, vectors comprising such polynucleotides, and the use of such polypeptides, polynucleotides and vectors for treating a disorder.

FIELD OF INVENTION

The present invention provides mutant polypeptides derived fromEscherichia coli dihydrofolate reductase (DHFR) which can be fused to apolypeptide of interest for efficient conditional modulation of itsactivity. Also disclosed are polynucleotides encoding such mutantpolypeptides, vectors comprising such polynucleotides, and the use ofsuch polypeptides, polynucleotides and vectors for treating a disorder.

BACKGROUND OF INVENTION

The rapidly changing field of gene therapy promises a number ofinnovative treatments for patients suffering from a variety of diseases.Advances in genetic modification of cancer and immune cells and the useof oncolytic viruses and bacteria have led to numerous clinical trialsfor cancer therapy, with several progressing to late-stage productdevelopment. Similarly, multiple clinical trials are underway testingthe efficacy of viral vectors (especially those based on recombinantadeno-associated viruses) for treatment of the diseases of the nervoussystem and eye diseases. At the time of this writing, very few genetherapy products have been approved by the United States Food and DrugAdministration (FDA) and the European Medicines Agency (EMA). Some ofthe key scientific and regulatory issues include understanding of genetransfer vector biology, safety of vectors in vitro and in animalmodels, optimum gene transfer, long-term persistence or integration inthe host, shedding of a virus and ability to maintain transgeneexpression in vivo for a desired period of time.

Techniques that target gene function at the level of DNA and mRNAprovide powerful methods for modulating the expression of proteinsencoded by specific genes. For example, the Cre/lox and tet/dox systemshave been widely used to target gene expression at the transcriptionallevel (Ryding et al., 2001) and RNA interference as a method to achievepost-transcriptional gene silencing (Fire et al., 1998; Madema, 2004).One of the major drawbacks of such methods in the context of genetherapy is that they often result in relatively high basal levels oftransgene even when not induced.

Methods for regulating protein activity, where the protein encoded bythe transgene is inactive or degraded in the absence of a specificligand, are promising alternatives.

However, methods for regulating protein function directly are limited,especially in mammalian cells. Inhibitors or activators of particularproteins have been identified, and often take the form of cell-permeablesmall molecules. Many of these molecules have found widespread use asbiological probes, often because of the speed, dosage-dependence, andreversibility of their activities, which complement methods formodulating gene expression at the DNA or RNA level. However theseinhibitors or activators are often promiscuous, affecting severalproteins rather than a specific protein.

Alternative strategies to perturb protein function by exploitingexisting cellular processes have been devised. For example, Varshayskyand coworkers developed methods for controlling protein function basedon the importance of certain N-terminal residues for protein stability(Bachmair et al., 1986). Szostak and coworkers showed that a smallpeptide sequence could be fused to the N-terminus of a protein ofinterest to modulate protein stability (Park et al., 1992). Varshayskyand coworkers have further isolated a temperature-sensitive peptidesequence that greatly reduced the half-life or dihydrofolate reductase(DHFR) at the non-permissive temperatures (Dohmen et al., 1994). Thisapproach has been used to study proteins in yeast (Labib et al., 2000;Kanemaki et al., 2003). More recently, several researchers haveengineered systems in which dimeric small molecules are used toconditionally target fusion proteins for degradation via E3 ligase orthe proteasome, itself (Schneekloth et al., 2004; Janse et al., 2004).However, these systems require either a prior knowledge of thehigh-affinity ligands that modulate the activity of a protein ofinterest or they are restricted to genetically engineered yeast strains.

An alternative approach for controlling protein function directly is tointerfere with subcellular localization. Several methods are availableto regulate protein localization using small-molecules by takingadvantage of the FKBP-rapamycin-FRB ternary complex.

U.S. application Ser. No. 12/069,235 describes mutant polypeptidesderived from Escherichia coli dihydrofolate reductase (DHFR) forconditionally controlling expression of a protein to which the mutantpolypeptides are fused. The resulting fusion protein is unstable and hasreduced activity in the absence of e.g. trimethoprim (TMP), which is aDHFR inhibitor. In the presence of TMP, the fusion protein is stabilisedand active.

In WO 2015/152813, this approach has been taken advantage of to developa system for conditionally stabilising a therapeutic protein, namely GTPcyclohydrolase 1 (GCH1) by fusion to a destabilising domain of DHFR. Theresulting fusion protein together with tyrosine hydrolase (TH) is usedfor gene therapy, where administration of a ligand such as TMP ensuresthat GCH1 is only fully active when the ligand is present.

There is however still a need for reliable, tunable and efficientmethods for conditionally stabilising a protein of interest, where thebasal activity level in the absence of ligand is even further reduced.

SUMMARY OF INVENTION

The present disclosure relates to mutant polypeptides derived fromEscherichia coli dihydrofolate reductase (DHFR) which can be fused to apolypeptide of interest for efficient conditional modulation of itsactivity. Also disclosed are polynucleotides encoding such mutantpolypeptides, vectors comprising such polynucleotides, and the use ofsuch polypeptides, polynucleotides and vectors for treating a disorder.

In a first aspect, the present disclosure relates to a mutantpolypeptide derived from DHFR comprising or consisting of a sequencediffering from SEQ ID NO: 1 and SEQ ID NO: 2 in at least one of thefollowing positions: W133, F153, R12, N18, M42, Y100, D122, P126 and/orD127, with the proviso that the mutant polypeptide does not differ fromSEQ ID NO: 1 and SEQ ID NO: 2 only by a Y100I mutation.

The mutations at positions W133, F153 share the common feature that theyresult in a particularly high degree of destabilization, which can befully recovered when TMP is applied as a ligand. This is evident fromTable 2 in the examples. Table 2 shows a very high fluorescence fromreporter protein YFP in the presence of TMP and a very low fluorescencewhen TMP is absent, i.e. when the protein is destabilized by the mutantDHFR.

In a further aspect, a fusion polypeptide comprising the mutantpolypeptide as described herein linked to a polypeptide of interest isprovided.

In yet a further aspect, is provided a polynucleotide encoding themutant polypeptide or the fusion polypeptide as described herein.

In yet a further aspect, a gene expression system comprising apolynucleotide as disclosed herein is provided.

Also provided is a system for conditionally stabilising a fusionpolypeptide comprising a polypeptide and a ligand as described herein,wherein the ligand is capable of binding to the polypeptide andstabilising the fusion polypeptide.

DESCRIPTION OF DRAWINGS

FIG. 1. Effect of loop structure variations on the reversibledestabilization of the DHFR domain fused to YFP reporter protein.Variations on W133, H141, and F153 were studied in the presence (blackbars) and absence (grey bars) of the TMP inhibitor and compared toreference construct containing the R12Y and Y100I variations (left mostpair of bars). Note that the YFP fluorescence is not dependent on thepresence of TMP when fused to the WT DHFR domain (right most pair ofbars).

FIG. 2. Effect of loop structure variations combined with Y100I, Y100Eor with other loop structure variations on the reversibledestabilization of the DHFR domain fused to YFP reporter protein.Variations were studied in the presence (black bars) and absence (greybars) of the TMP inhibitor and compared to reference constructcontaining the R12Y and Y100I variations (left most pair of bars). Notethat the scale on the Y axis is different from the scale in FIG. 1. WhenW133 or F153 variants with reversible destabilization characteristicsare combined with either of the Y100I or Y100E variation, the resultingcombined variants are no longer rescuable with TMP. The same result wasobtained when W133 and F153 variations were combined together.

DEFINITIONS

The term “system”, as used herein, denotes a system specificallydesigned for production of a specific gene product, which in this caseis a fusion protein, as specified in the claims and explained in furtherdetail below. The gene expression system may be used in vitro, but inmany embodiments it is intended to be used in gene therapy, both in vivoand ex vivo.

In the context of the present disclosure, the terms “polypeptide” and“protein” are used interchangeably. They both relate to a compoundconsisting of a contiguous sequence of amino acid residues linked bypeptide bonds.

Furthermore, in the context of polypeptides discussed herein, the term“based on” may be used interchangeably with the term “derived from”. Apolypeptide or peptide, or fragment thereof, derived from a specificpolypeptide or peptide (“parent polypeptide”) has an amino acid sequencethat is homologous to but not 100% identical with the parentpolypeptide. A derivative is thus a polypeptide or peptide, or fragmentthereof, derived from a parent polypeptide. An analogue is such aderivative that has essentially the same function or exactly the samefunction as the parent polypeptide.

In the context of polypeptides discussed herein, the term “variant” maybe used interchangeably with the term “derived from”. The variant may bea polypeptide having an amino acid sequence that does not occur innature.

A mutant polypeptide or a mutated polypeptide is a polypeptide that hasbeen designed or engineered in order to alter the properties of a parentpolypeptide. A mutant polypeptide derived from DHFR and comprising orconsisting of a sequence differing from a parent sequence in at leastone position refers to a polypeptide having essentially the samesequence as the parent sequence except in the indicated positions. Byway of example, a mutant polypeptide derived from DHFR and comprising orconsisting of a sequence differing from SEQ ID NO: 1 at least inposition R12 and having at least 70% identity to SEQ ID NO: 1 has asequence which is at least 70% identical to SEQ ID NO: 1, wherein theresidue in position 12 is not an R residue.

The term “nucleotide sequence” as used herein may also be denoted by theterm “nucleic acid sequence”. Below, the expression “gene” is sometimesused, which is a nucleotide sequence encoding for a polypeptide.

The term “polynucleotide” as used herein refers to a compound consistingof a contiguous sequence of nucleotides linked by covalent bonds.

The term “fusion polypeptide” as used herein relates to a polypeptideobtained after fusion of two or more polypeptides, i.e. arrangementin-frame as part of the same contiguous sequence of amino acid residues.Fusion can be direct, i.e. with no additional amino acid residuesbetween the two polypeptides, or achieved via a linker. Such a linkermay be used to improve performance and/or alter the functionality.

The term “domain” as used herein, which may also be denoted “region”,refers to a contiguous sequence of amino acid residues that has aspecific function, such as binding to a ligand and/or conferringinstability, or to a contiguous sequence of nucleotides coding for acontiguous sequence of amino acid residues that has a specific function.The term “domain” may also refer to an independently folding unit withina protein.

The term “gene therapy” as used herein refers to the insertion of genesinto a subject's cells and tissues to treat a disease.

The term “therapeutic” in relation to a polypeptide, a protein or apolynucleotide refers to the ability of the polypeptide, the protein orthe polypeptide or protein encoded by the polynucleotide to exert atherapeutic activity.

The term “operatively linked” as used herein refers to a mutantpolypeptide and a polypeptide of interest linked in such a manner thatwhen the mutant polypeptide is stable and thus active, i.e. in thepresence of a ligand as described herein, the polypeptide of interest isalso stable and active, while in the absence of a ligand, thedestabilisation of the mutant polypeptide leads also to thedestabilisation of the polypeptide of interest.

Throughout the present disclosure, mutations will be designated asfollows: XNZ, where X is the one-letter amino acid code for the aminoacid present in the DHFR sequence(s) from Escherichia coli as set forthin SEQ ID NO: 1 at position N, while Z is the one-letter amino acid codefor the amino acid substituting X at position N in the mutant.

DETAILED DESCRIPTION OF THE INVENTION

The invention is as described in the claims.

The present disclosure relates to a method for conditionally stabilisinga fusion polypeptide. The fusion polypeptide is a fusion of a mutantpolypeptide derived from DHFR and of a polypeptide of interest, of whichit is desirable to tightly control the activity. The mutant polypeptidecan be bound by a DHFR inhibitor. In the absence of the inhibitor, themutant polypeptide, and consequently also the fusion polypeptide and thepolypeptide of interest, is not active. In the presence of DHFRinhibitor, the mutant polypeptide is bound by the inhibitor, whichbinding results in stabilisation of the mutant polypeptide and thus alsoof the fusion polypeptide and of the polypeptide of interest. As aconsequence, the polypeptide of interest is active and/or functional inthe presence of ligand only. This system can be used as a safety switchin gene therapy, where the administration of a ligand is necessary forthe transgene to lead to a functional and/or active protein. In theabsence of ligand, the transgene is expressed but leads to an inactiveprotein. In some embodiments, the present system has a dose-dependentresponse, and can thus be taken advantage of to precisely tune theextent of activation of the polypeptide of interest, by adjusting theamount of ligand. This can be desirable in order to personalise atreatment depending on the subject, the disorder to be treated, thenature of the polypeptide of interest, or the need to vary the dose of apolypeptide of interest over time.

DHFR is a 159-residue enzyme catalyzing the reduction of dihydrofolateto tetrahydrofolate, a cofactor that is essential for several steps inprokaryotic primary metabolism (Schnell et al., 2004). Unless otherwisespecified, the term “DHFR” as used herein refers to E. coli DHFR.

Numerous inhibitors of DHFR have been developed as drugs (Schweitzer etal., 1990), and one such inhibitor, trimethoprim (TMP), inhibits DHFRfrom E. coli (ecDHFR) much more potently than mammalian DHFR (Matthewset al., 1985). This large therapeutic window renders TMP “biologicallysilent” in mammalian cells, which is particularly relevant in thecontext of gene therapy. The specificity of the ecDHFR-TMP interaction,coupled with the commercial availability and attractive pharmacologicalproperties of TMP, makes this protein-ligand pair ideal for developmentof a conditional protein stability system as described herein.

Mutant Polypeptide Derived from DHFR

The present disclosure provides mutant polypeptides derived from DHFR asset forth in SEQ ID NO: 1 (corresponding to wild type E. coli DHFR) andin SEQ ID NO: 2 in at least one of the following positions: R12, N18,M42, Y100, D122, P126, D127, W133 and/or F153, with the proviso that themutant polypeptide does not differ from SEQ ID NO: 1 and SEQ ID NO: 2only by a Y100I mutation. As will be seen from the examples, such mutantpolypeptides are particularly advantageous in conditional proteinstability systems and have applications e.g. in gene therapy. The mutantpolypeptides can be fused to a protein of interest, of which theactivity can then be modulated by the presence or absence of a ligand,e.g. a DHFR inhibitor. The mutant polypeptides provided herein have theability to allow for sufficient stability, and hence activity, of theprotein of interest in the presence of a DHFR inhibitor, while at thesame time efficiently shutting down the protein's activity in theabsence of DHFR inhibitor.

In some embodiments, the mutant polypeptide derived from DHFR as setforth in SEQ ID NO: 1 and SEQ ID NO: 2 has at least 70% identity to SEQID NO: 1, such as at least 75% identity, such as 80% identity, such asat least 85% identity, such as at least 90% identity, such as at least95% identity, such as at least 96% identity, such as at least 97%identity, such as at least 98% identity, such as at least 99% identityto SEQ ID NO: 1. In other embodiments, the mutant polypeptide derivedfrom DHFR as set forth in SEQ ID NO: 1 and SEQ ID NO: 2 has at least 70%identity to SEQ ID NO: 2, such as at least 75% identity, such as 80%identity, such as at least 85% identity, such as at least 90% identity,such as at least 95% identity, such as at least 96% identity, such as atleast 97% identity, such as at least 98% identity, such as at least 99%identity to SEQ ID NO: 2.

In some embodiments, the mutant polypeptide differs from DHFR as setforth in SEQ ID NO: 1 by the presence of at least one mutation inposition R12 and has at least 70% identity to SEQ ID NO: 1, such as atleast 75% identity, such as at least 80% identity, such as at least 85%identity, such as at least 90% identity, such as at least 95% identity,such as at least 96% identity, such as at least 97% identity, such as atleast 98% identity, such as at least 99% identity to SEQ ID NO: 1. Insome embodiments, the mutant polypeptide differs from DHFR as set forthin SEQ ID NO: 1 by the presence of at least one mutation in position N18and has at least 70% identity to SEQ ID NO: 1, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 1. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 by the presence of at least one mutation in position M42and has at least 70% identity to SEQ ID NO: 1, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 1. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 by the presence of at least one mutation in position Y100,and has at least 70% identity to SEQ ID NO: 1, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 1. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 by the presence of at least one mutation in position D122and has at least 70% identity to SEQ ID NO: 1, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 1. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 by the presence of at least one mutation in position P126and has at least 70% identity to SEQ ID NO: 1, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 1. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 by the presence of at least one mutation in position D127and has at least 70% identity to SEQ ID NO: 1, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 1. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 by the presence of at least one mutation in position W133and has at least 70% identity to SEQ ID NO: 1, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 1. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 by the presence of at least one mutation in position F153and has at least 70% identity to SEQ ID NO: 1, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 1. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 by the presence of at least one mutation in a positionselected from the group consisting of W133, F153 or F153 and has atleast 70% identity to SEQ ID NO: 1, such as at least 75% identity, suchas at least 80% identity, such as at least 85% identity, such as atleast 90% identity, such as at least 95% identity, such as at least 96%identity, such as at least 97% identity, such as at least 98% identity,such as at least 99% identity to SEQ ID NO: 1.

In some embodiments, the mutant polypeptide differs from DHFR as setforth in SEQ ID NO: 2 by the presence of at least one mutation inposition R12 and has at least 70% identity to SEQ ID NO: 2, such as atleast 75% identity, such as at least 80% identity, such as at least 85%identity, such as at least 90% identity, such as at least 95% identity,such as at least 96% identity, such as at least 97% identity, such as atleast 98% identity, such as at least 99% identity to SEQ ID NO: 2. Insome embodiments, the mutant polypeptide differs from DHFR as set forthin SEQ ID NO: 2 by the presence of at least one mutation in position N18and has at least 70% identity to SEQ ID NO: 2, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 2. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 2 by the presence of at least one mutation in position M42and has at least 70% identity to SEQ ID NO: 2, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 2. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 2 by the presence of at least one mutation in position Y100,and has at least 70% identity to SEQ ID NO: 2, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 2. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 2 by the presence of at least one mutation in position D122and has at least 70% identity to SEQ ID NO: 2, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 2. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 2 by the presence of at least one mutation in position P126and has at least 70% identity to SEQ ID NO: 2, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 2. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 2 by the presence of at least one mutation in position D127and has at least 70% identity to SEQ ID NO: 2, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 2. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 2 by the presence of at least one mutation in position W133and has at least 70% identity to SEQ ID NO: 2, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 2. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 2 by the presence of at least one mutation in position F153and has at least 70% identity to SEQ ID NO: 2, such as at least 75%identity, such as at least 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 2. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 2 by the presence of at least one mutation in a positionselected from the group consisting of W133, F153 or F153 and has atleast 70% identity to SEQ ID NO: 2, such as at least 75% identity, suchas at least 80% identity, such as at least 85% identity, such as atleast 90% identity, such as at least 95% identity, such as at least 96%identity, such as at least 97% identity, such as at least 98% identity,such as at least 99% identity to SEQ ID NO: 2.

In some embodiments, the mutant polypeptide differs from DHFR as setforth in SEQ ID NO: 1 and in SEQ ID NO: 2 by the presence of at leastone mutation in position R12. In some embodiments, the mutantpolypeptide differs from DHFR as set forth in SEQ ID NO: 1 and in SEQ IDNO: 2 by the presence of at least one mutation in position N18. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 and in SEQ ID NO: 2 by the presence of at least onemutation in position M42. In some embodiments, the mutant polypeptidediffers from DHFR as set forth in SEQ ID NO: 1 and in SEQ ID NO: 2 bythe presence of at least one mutation in position Y100. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 and in SEQ ID NO: 2 by the presence of at least onemutation in position D122. In some embodiments, the mutant polypeptidediffers from DHFR as set forth in SEQ ID NO: 1 and in SEQ ID NO: 2 bythe presence of at least one mutation in position P126. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 and in SEQ ID NO: 2 by the presence of at least onemutation in position D127. In some embodiments, the mutant polypeptidediffers from DHFR as set forth in SEQ ID NO: 1 and in SEQ ID NO: 2 bythe presence of at least one mutation in position W133. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 and in SEQ ID NO: 2 by the presence of at least onemutation in position F153. In some embodiments, the mutant polypeptidediffers from DHFR as set forth in SEQ ID NO: 1 and in SEQ ID NO: 2 bythe presence of at least one mutation in a position selected from thegroup consisting of W133, F153 or F153.

In some embodiments, the mutant polypeptide differs from DHFR as setforth in SEQ ID NO: 1 and in SEQ ID NO: 2 by the presence of at least anR12A mutation. In some embodiments, the mutant polypeptide differs fromDHFR as set forth in SEQ ID NO: 1 and in SEQ ID NO: 2 by the presence ofat least an N18T mutation. In some embodiments, the mutant polypeptidediffers from DHFR as set forth in SEQ ID NO: 1 and in SEQ ID NO: 2 bythe presence of at least an M42A mutation. In some embodiments, themutant polypeptide differs from DHFR as set forth in SEQ ID NO: 1 and inSEQ ID NO: 2 by the presence of at least an M42G mutation. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 and in SEQ ID NO: 2 by the presence of at least a Y100Emutation. In some embodiments, the mutant polypeptide differs from DHFRas set forth in SEQ ID NO: 1 and in SEQ ID NO: 2 by the presence of atleast a D122A mutation. In some embodiments, the mutant polypeptidediffers from DHFR as set forth in SEQ ID NO: 1 and in SEQ ID NO: 2 bythe presence of at least a P126Y mutation. In some embodiments, themutant polypeptide differs from DHFR as set forth in SEQ ID NO: 1 and inSEQ ID NO: 2 by the presence of at least a P126R mutation. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 and in SEQ ID NO: 2 by the presence of at least a P126Dmutation. In some embodiments, the mutant polypeptide differs from DHFRas set forth in SEQ ID NO: 1 and in SEQ ID NO: 2 by the presence of atleast a D127A mutation. In some embodiments, the mutant polypeptidediffers from DHFR as set forth in SEQ ID NO: 1 and in SEQ ID NO: 2 bythe presence of at least a D127N mutation. In some embodiments, themutant polypeptide differs from DHFR as set forth in SEQ ID NO: 1 and inSEQ ID NO: 2 by the presence of at least a W133A mutation. In someembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 and in SEQ ID NO: 2 by the presence of at least an F153Gmutation. In some embodiments, the mutant polypeptide differs from DHFRas set forth in SEQ ID NO: 1 and in SEQ ID NO: 2 by the presence of atleast an F153A mutation. In some embodiments, the mutant polypeptidediffers from DHFR as set forth in SEQ ID NO: 1 and in SEQ ID NO: 2 bythe presence of at least an F153D mutation. In some preferredembodiments, the mutant polypeptide differs from DHFR as set forth inSEQ ID NO: 1 and in SEQ ID NO: 2 by the presence of at least onemutation selected from the group consisting of W133A, F153G or F153D.

In some embodiments, the mutant polypeptide does not differ from SEQ IDNO: 1 and SEQ ID NO: 2 solely by the presence of a Y100I mutation.

In one embodiment, the mutant polypeptide is an R12A mutant. In anotherembodiment, the mutant polypeptide is an N18T mutant. In anotherembodiment, the mutant polypeptide is an M42A mutant. In anotherembodiment, the mutant polypeptide is an M42G mutant. In anotherembodiment, the mutant polypeptide is a Y100E mutant. In anotherembodiment, the mutant polypeptide is a D122A mutant. In anotherembodiment, the mutant polypeptide is a P126Y mutant. In anotherembodiment, the mutant polypeptide is a P126D mutant. In anotherembodiment, the mutant polypeptide is a P126R mutant. In anotherembodiment, the mutant polypeptide is a D127A mutant. In anotherembodiment, the mutant polypeptide is a D127N mutant. In anotherembodiment, the mutant polypeptide is a W133A mutant. In anotherembodiment, the mutant polypeptide is an F153G mutant. In anotherembodiment, the mutant polypeptide is an F153A mutant. In anotherembodiment, the mutant polypeptide is an F153D mutant. In particularembodiments, the mutant polypeptide is an M42A mutant, an M42G mutant, aY100E mutant, a P126Y mutant, a P126R mutant, a P126D mutant, a W133Amutant, an F153G mutant, an F153A mutant or an F153D mutant.

In some embodiments, the mutant polypeptide comprises two mutations. Inone embodiment, the mutant polypeptide is an R12A Y100I mutant. Inanother embodiment, the mutant is an N18T Y100I mutant. In anotherembodiment, the mutant polypeptide is a Y100I D122A mutant. In anotherembodiment, the mutant polypeptide is a Y100I D127N mutant. In anotherembodiment, the mutant polypeptide is a Y100I W133A mutant. In anotherembodiment, the mutant polypeptide is a Y100I F153D mutant. In anotherembodiment, the mutant polypeptide is a Y100I F153G mutant. In anotherembodiment, the mutant polypeptide is an R12A Y100E mutant. In anotherembodiment, the mutant polypeptide is an N18T Y100E mutant. In anotherembodiment, the mutant polypeptide is an M42A Y100E mutant. In anotherembodiment, the mutant polypeptide is an M42G Y100E mutant. In anotherembodiment, the mutant polypeptide is a Y100E D127A mutant. In anotherembodiment, the mutant polypeptide is a Y100E D127N mutant. In anotherembodiment, the mutant polypeptide is a Y100E P126Y mutant. In anotherembodiment, the mutant polypeptide is a Y100E P126D mutant. In anotherembodiment, the mutant polypeptide is a Y100E P126R mutant. In anotherembodiment, the mutant polypeptide is a Y100E W133A mutant. In anotherembodiment, the mutant polypeptide is a Y100E F153D mutant. In anotherembodiment, the mutant polypeptide is a Y100E F153G mutant. Inparticular embodiments, the mutant polypeptide is an N18T Y100I mutant,a Y100I D127N mutant, or an M42A Y100E mutant.

In some embodiments, the mutant polypeptide comprises three mutations.In one embodiment, the mutant polypeptide is an N18T Y100I D122A mutant.In another embodiment, the mutant polypeptide is an N18T Y100E D122Amutant.

In some embodiments, the mutant polypeptide does not differ from theparent sequence by more than five residues, such as by more than fourresidues, such as by more than three residues, such as by more than tworesidues, such as by more than one residue. Some mutant polypeptides ofthe present disclosure do not differ from SEQ ID NO: 1 by more than fiveresidues. Other mutant polypeptides of the present disclosure do notdiffer from SEQ ID NO: 1 by more than four residues. Other mutantpolypeptides of the present disclosure do not differ from SEQ ID NO: 1by more than three residues. Other mutant polypeptides of the presentdisclosure do not differ from SEQ ID NO: 1 by more than two residues.Other mutant polypeptides of the present disclosure do not differ fromSEQ ID NO: 1 by more than one residue. Preferably, the mutantpolypeptide does not differ from SEQ ID NO: 1 solely by a Y100Imutation. Some mutant polypeptides of the present disclosure do notdiffer from SEQ ID NO: 2 by more than five residues. Other mutantpolypeptides of the present disclosure do not differ from SEQ ID NO: 2by more than four residues. Other mutant polypeptides of the presentdisclosure do not differ from SEQ ID NO: 2 by more than three residues.Other mutant polypeptides of the present disclosure do not differ fromSEQ ID NO: 2 by more than two residues. Other mutant polypeptides of thepresent disclosure do not differ from SEQ ID NO: 2 by more than oneresidue. Preferably, the mutant polypeptide does not differ from SEQ IDNO: 2 solely by a Y100I mutation.

Besides the specific embodiments listed herein above, the mutantpolypeptide may comprise further mutations as described below.

Partial or Total Removal of Side Chain at a Given Position

Without being bound by theory, some mutations in the above-mentionedpositions which replace an amino acid having a side chain with an aminoacid having a shorter side chain or no side chain appear particularlyadvantageous. Accordingly, in some embodiments the mutant polypeptidediffers from SEQ ID NO: 1 and SEQ ID NO:2 by the presence of a mutationin at least one of R12, N18, M42, Y100, D122, P126, D127, W133 and/orF153, wherein the mutation is a substitution by a G residue or by an Aresidue. Substitution by a glycine residue completely removes the sidechain at a given position, while substitution by an alanine residuepartly removes or shortens the side chain at a given position.

Hence, in some preferred embodiments, the mutant polypeptide differsfrom DHFR as set forth in SEQ ID NO: 1 and in SEQ ID NO: 2 by thepresence of one mutation selected from the group consisting of R12A,R12G, N18A, N18G, M42A, M42G, D122A, D122G, P126A, P126G, D127A, D127G,W133A, W133G, F153A and F153G, preferably W133A, F153G, R12A, M42A orF153A.

Additional Mutations

The mutant polypeptide may further comprise one or more additionalmutations. These may confer further advantageous properties to thefusion polypeptides comprising the mutant polypeptide fused to apolypeptide of interest as described herein below.

Thus in some embodiments, the mutant polypeptide differs from SEQ ID NO:1 and SEQ ID NO: 2 in at least one of positions R12, N18, M42, Y100,D122, P126, D127, W133 and/or F153, and in a further position.

In some embodiments, the mutant polypeptide further comprises a mutationat position Y100, such as Y100E or Y100I, preferably Y100E. In someembodiments, the mutant polypeptide is an M42A Y100E mutant. In someembodiments, the mutant polypeptide is an N18T Y100I mutant. In someembodiments, the mutant is a D127N Y100I mutant.

Further mutations located in the TMP binding pocket of DHFR may also beadvantageous. The TMP binding pocket is the pocket formed by DHFR whichinteracts with TMP. The following residues are known to be involved(Roth et al., 1987, J Med Chem. 1987 February; 30(2):348-56): M20, L28,F31, T46, S49, 150 and L54.

Polynucleotides and Vectors

Also provided herein are polynucleotides encoding any of the mutantpolypeptides described herein above, as well as vectors comprising suchpolynucleotides. Such vectors may comprise several mutant polypeptidesas described herein.

Fusion Polypeptides

The mutant polypeptides described herein above are useful in fusionpolypeptides, where they are operatively linked to one or morepolypeptides of interest. In the absence of ligand, the mutantpolypeptide is unstable, and this in turn results in the polypeptide ofinterest being unstable and hence inactive. This is the “off”configuration. In the presence of ligand, the mutant polypeptide isstable, and the polypeptide of interest is also stable and hence active.The addition of ligand can thus be used as safety switch preventing theunwanted activation of a polypeptide of interest, e.g. in the context ofgene therapy, in the absence of ligand.

The present disclosure thus provides fusion polypeptides comprising amutant polypeptide as described herein operatively linked to apolypeptide of interest. The mutant polypeptides are derived from DHFRas set forth in SEQ ID NO: 1 (corresponding to wild type E. coli DHFR)and in SEQ ID NO: 2 in at least one of the following positions: R12,N18, M42, Y100, D122, P126, D127, W133 and/or F153, with the provisothat the mutant polypeptide does not differ from SEQ ID NO: 1 and SEQ IDNO: 2 only by a Y100I mutation, as described herein above. In someembodiments, the mutant polypeptide derived from DHFR as set forth inSEQ ID NO: 1 and SEQ ID NO: 2 has at least 70% identity to SEQ ID NO: 1,such as at least 75% identity, such as 80% identity, such as at least85% identity, such as at least 90% identity, such as at least 95%identity, such as at least 96% identity, such as at least 97% identity,such as at least 98% identity, such as at least 99% identity to SEQ IDNO: 1. In other embodiments, the mutant polypeptide derived from DHFR asset forth in SEQ ID NO: 1 and SEQ ID NO: 2 has at least 70% identity toSEQ ID NO: 2, such as at least 75% identity, such as 80% identity, suchas at least 85% identity, such as at least 90% identity, such as atleast 95% identity, such as at least 96% identity, such as at least 97%identity, such as at least 98% identity, such as at least 99% identityto SEQ ID NO: 2.

In some embodiments, it may be desirable to be able to monitor theactual expression of the fusion polypeptides. Accordingly, in someembodiments, the fusion polypeptide may also comprise a reporterpolypeptide. The reporter polypeptide may be operatively linked to themutant polypeptide or to the polypeptide of interest by one of itstermini, or it may be linked to both the mutant polypeptide and to thepolypeptide of interest, at each of its termini.

The reporter polypeptide may be any reporter polypeptide known in theart and suitable for monitoring purposes. In some embodiments, thereporter polypeptide is a fluorescent protein, of which theactivity/stability can be monitored by fluorescence microscopy methodsas is known to the person of skill in the art. In other embodiments, thereporter polypeptide is an enzyme, of which the activity can be measuredby enzymatic assays. In yet other embodiments, the reporter polypeptideis an affinity tag, of which the activity can be measured as is known inthe art.

Preferably, the fusion polypeptides described herein are such that themutant polypeptide is operatively linked to the N-terminal end of thepolypeptide of interest. However, fusion polypeptides where the mutantpolypeptide is further operatively linked to the C-terminal end of asecond or additional polypeptide of interest are also envisaged.

Accordingly, in some embodiments, the mutant polypeptide may beoperatively linked to the N-terminal end of a polypeptide of interestand may be operatively further linked to the C-terminal end of a furtherpolypeptide of interest. Such fusion polypeptides thus may allow forconditional stability of two polypeptides of interest at once.

It will be understood that the polypeptide of interest or the furtherpolypeptide of interest linked to the C-terminal end or the N-terminalend of the mutant polypeptide may itself further be operatively linkedto a further polypeptide of interest or to a reporter polypeptide in itsother terminal end.

The person of skill in the art knows how to design linkers that aresuitable for operatively linking the polypeptide of interest to themutant polypeptide.

Polypeptide of Interest

Any of the above mentioned polypeptides of interest, i.e. thepolypeptide of interest to which the mutant polypeptide is fused, thefurther polypeptide of interest or any additional polypeptide ofinterest, may be any polypeptide for which it is desirable toconditionally control the stability, and hence activity, e.g. in a hostcell. Preferably, the polypeptide of interest is a therapeuticpolypeptide, a reporter polypeptide, an enzyme or a transcriptionfactor.

Non-limiting examples of therapeutic polypeptides include transcriptionfactors, neurotrophic factors, cell surface receptors, ATPases,cyclin-dependent kinase inhibitors and antibodies having a therapeuticeffect when active in a host cell, e.g. of a subject in need oftreatment for a disorder.

Additional Features

The fusion polypeptides described herein may further comprise additionalfeatures. In some cases, it may be desirable to include a signalpeptide, e.g. a signal peptide capable of causing secretion of thefusion polypeptide from a cell. This would allow for secretion of apolypeptide of interest in a recombinant host, e.g. a microbial cellsuch as a yeast cell or a bacterial cell. It may be desirable to includea cleavage signal so that only the polypeptide of interest, without themutant polypeptide, is secreted, or so that the polypeptide of interestcan be isolated without being fused to the mutant polypeptide. In othercases, it may be relevant to include a subcellular localisation signalsuch as a nuclear localisation signal capable of causing import of thefusion polypeptide into the nucleus or some other compartment of a cell;this may be particularly relevant if the polypeptide of interest is atranscription factor. The skilled person knows how to design a fusionpolypeptide so that it comprises such signal peptides.

Polynucleotides and Vectors

Also provided herein are polynucleotides encoding any of the fusionpolypeptides described herein above, as well as vectors comprising suchpolynucleotides. Such vectors may comprise several fusion polypeptidesas described herein.

Gene Expression System

Also provided herein is a gene expression system comprising apolynucleotide encoding a fusion polypeptide as described herein. Thegene expression system of the present invention may comprise a vectorcomprising a polynucleotide encoding a fusion polypeptide as describedherein. In some embodiments, the gene expression system is suitable foradministration in the context of gene therapy to a subject in needthereof.

Broadly, gene therapy seeks to transfer new genetic material to thecells of a patient with resulting therapeutic benefit to the patient.Such benefits include treatment or prophylaxis of a broad range ofdiseases, disorders and other conditions.

Ex vivo gene therapy approaches involve modification of isolated cells(including but not limited to stem cells, neural and glial precursorcells, and fetal stem cells), which are then infused, grafted orotherwise transplanted into the patient. See, e.g., U.S. Pat. Nos.4,868,116, 5,399,346 and 5,460,959. In vivo gene therapy seeks todirectly target host patient tissue in vivo.

Viruses useful as gene transfer vectors include papovavirus, adenovirus,vaccinia virus, adeno-associated virus, herpesvirus, and retroviruses.Suitable retroviruses include the group consisting of HIV, SIV, FIV,EIAV, MoMLV. A further group of suitable retroviruses includes the groupconsisting of HIV, SIV, FIV, EAIV, CIV. Another group of preferred virusvectors includes the group consisting of alphavirus, adenovirus, adenoassociated virus, baculovirus, HSV, coronavirus, Bovine papilloma virus,Mo-MLV, preferably adeno associated virus (AAV).

Lentiviruses and adeno-associated viruses are preferred vectors for usein the treatment of disorders of the central nervous system. Both typesof viruses can integrate into the genome without cell divisions, andboth types have been tested in pre-clinical animal studies forindications of the nervous system, in particular the central nervoussystem.

Methods for preparation of AAV are described in the art, e.g. U.S. Pat.No. 5,677,158. U.S. Pat. Nos. 6,309,634 and 6,683,058 describe examplesof delivery of AAV to the central nervous system.

A lentiviral vector is a replication-defective lentivirus particle. Sucha lentivirus particle can be produced from a lentiviral vectorcomprising a 5′ lentiviral LTR, a tRNA binding site, a packaging signal,a promoter operably linked to a polynucleotide signal encoding saidfusion protein, an origin of second strand DNA synthesis and a 3′lentiviral LTR. Methods for preparation and in vivo administration oflentivirus to neural cells are described in US 20020037281 (Methods fortransducing neural cells using lentiviral vectors).

Retroviral vectors are the vectors most commonly used in human clinicaltrials, since they can carry 7-8 kb or heterologous DNA and since theyhave the ability to infect cells and have their genetic material stablyintegrated into the host cell with high efficiency. See, e.g., WO95/30761; WO 95/24929. Oncovirinae require at least one round of targetcell proliferation for transfer and integration of exogenous nucleicacid sequences into the patient. Retroviral vectors integrate randomlyinto the patient's genome. Retroviruses can be used to target stem cellsof the nervous system as very few cell divisions take place in othercells of the nervous system (in particular the CNS).

Three classes of retroviral particles have been described; ecotropic,which can infect murine cells efficiently, and amphotropic, which caninfect cells of many species. The third class includes xenotrophicretrovirus which can infect cells of another species than the specieswhich produced the virus. Their ability to integrate only into thegenome of dividing cells has made retroviruses attractive for markingcell lineages in developmental studies and for delivering therapeutic orsuicide genes to cancers or tumors.

For use in human patients, the retroviral vectors must be replicationdefective. This prevents further generation of infectious retroviralparticles in the target tissue—instead the replication defective vectorbecomes a “captive” transgene stable incorporated into the target cellgenome. Typically in replication defective vectors, the gag, env, andpol genes have been deleted (along with most of the rest of the viralgenome). Heterologous DNA is inserted in place of the deleted viralgenes. The heterologous genes may be under the control of the endogenousheterologous promoter, another heterologous promoter active in thetarget cell, or the retroviral 5′ LTR (the viral LTR is active indiverse tissues). Typically, retroviral vectors have a transgenecapacity of about 7-8 kb.

Replication defective retroviral vectors require provision of the viralproteins necessary for replication and assembly in trans, from, e.g.,engineered packaging cell lines. It is important that the packagingcells do not release replication competent virus and/or helper virus.This has been achieved by expressing viral proteins from RNAs lackingthe ψ signal, and expressing the gag/pol genes and the env gene fromseparate transcriptional units. In addition, in some retroviruses ofsecond or third generation, the 5′ LTR's have been replaced withnon-viral promoters controlling the expression of these genes, and the3′ promoter has been minimised to contain only the proximal promoter.These designs minimize the possibility of recombination leading toproduction of replication competent vectors, or helper viruses.

Construction of vectors for use in the methods disclosed herein may beaccomplished using conventional techniques which do not require detailedexplanation to one of ordinary skill in the art. For review, however,those of ordinary skill may wish to consult Maniatis et al., inMolecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,(NY 1982). Expression vectors may be used for generating producer cellsfor recombinant production of a polypeptide of interest such as atherapeutic peptide, for example for medical use, and for generatingtherapeutic cells secreting a polypeptide of interest for naked orencapsulated therapy.

Briefly, construction of recombinant expression vectors employs standardligation techniques. For analysis to confirm correct sequences invectors constructed, the genes are sequenced using, for example, Sangersequencing or next generation sequencing or other suitable methods whichwill be known to those skilled in the art.

Size separation of cleaved fragments is performed using conventional gelelectrophoresis as described, for example, by Maniatis, et al.,(Molecular Cloning, pp. 133-134, 1982).

For generation of efficient expression vectors, these should containregulatory sequences necessary for expression of the encoded gene in thecorrect reading frame. Expression of a gene is controlled at thetranscription, translation or post-translation levels. Transcriptioninitiation is an early and critical event in gene expression. Thisdepends on the promoter and enhancer sequences and is influenced byspecific cellular factors that interact with these sequences. Thetranscriptional unit of many genes consists of the promoter and in somecases enhancer or regulator elements (Banerji et al., Cell 27: 299(1981); Corden et al., Science 209: 1406 (1980); and Breathnach andChambon, Ann. Rev. Biochem. 50: 349 (1981)). For retroviruses, controlelements involved in the replication of the retroviral genome reside inthe long terminal repeat (LTR) (Weiss et al., eds., The molecularbiology of tumor viruses: RNA tumor viruses, Cold Spring HarborLaboratory, (NY 1982)). Moloney murine leukemia virus (MLV) and Roussarcoma virus (RSV) LTRs contain promoter and enhancer sequences (Jollyet al., Nucleic Acids Res. 11: 1855 (1983); Capecchi et al., In:Enhancer and eukaryotic gene expression, Gulzman and Shenk, eds., pp.101-102, Cold Spring Harbor Laboratories (NY 1991). Other potentpromoters include those derived from cytomegalovirus (CMV) and otherwild-type viral promoters.

Promoter and enhancer regions of a number of non-viral promoters havealso been described (Schmidt et al., Nature 314: 285 (1985); Rossi anddeCrombrugghe, Proc. Natl. Acad. Sci. USA 84: 5590-5594 (1987)). Methodsfor maintaining and increasing expression of transgenes in quiescentcells include the use of promoters including collagen type I (1 and 2)(Prockop and Kivirikko, N. Eng. J. Med. 311: 376 (1984); Smith andNiles, Biochem. 19: 1820 (1980); de Wet et al., J. Biol. Chem., 258:14385 (1983)), SV40 and LTR promoters. Promoters and other regulatoryelements of the present invention are described in further detail hereinbelow.

In one embodiment the expression system is a vector, such as a viralvector, e.g. a viral vector expression system.

In another embodiment, the expression system is a plasmid vectorexpression system.

In yet another embodiment the expression system is based on a syntheticvector.

In yet another embodiment the expression system is a cosmid vector or anartificial chromosome.

In one embodiment the expression system according to the presentdisclosure is a viral vector selected from the group consisting of anadeno associated vector (AAV), adenoviral vector and retroviral vector.

In one embodiment the vector is an integrating vector. In anotherembodiment the vector is a non-integrating vector.

In one embodiment the vector of the present disclosure is a minimallyintegrating vector.

In one embodiment the expression system according to the presentdisclosure is an adeno associated vector (AAV).

AAV vectors can be prepared using two major principles, transfection ofhuman cell line monolayer culture or free floating insect cells.Monolayer cell cultures are transfected through calcium phosphateprecipitation, lipofection or other means with a mix of two or threeplasmid preparations containing a transfer plasmid with the vectorgenome and one or two helper plasmids containing the necessary genes forvector capsid synthesis. For insect cell cultures, this process isnormally replaced by transfection of the cells using baculovirusconstructs that contain the same functions. The cells, supernatant orboth are then collected for purification and concentration of thevector. This can be achieved through any combination of caesium chlorideor iodixanol gradient purification, ion exchange chromatography, gelfiltration and affinity chromatography and ultracentrifugation. Methodsfor preparation of AAV are described in the art, e.g. U.S. Pat. Nos.5,677,158, 6,309,634, and 6,451,306 describe examples of delivery of AAVto the central nervous system.

The AAV vector may be of any serotype selected to have specificity for agiven target cell. In one embodiment the AAV vector according to thepresent invention is selected from the group consisting of serotypesAAV5, AAV1, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV2 vectors. Furtherincluded within the scope of the application are recombinant (synthetic)AAV vectors engineered to have a particular cellular specificity and thenon-human primate equivalents of AAV.

In one embodiment the vector according to the present invention iscapable of infecting or transducing mammalian cells.

System for Conditionally Stabilising a Fusion Polypeptide

The present fusion polypeptides, polynucleotides, vectors and geneexpression systems are useful for conditionally stabilising a fusionpolypeptide. Thus are also provided herein a system for conditionallystabilising a fusion polypeptide, said system comprising a fusionpolypeptide, a polynucleotide encoding a fusion polypeptide, a vectorcomprising such a polynucleotide or a gene expression system asdescribed herein above, and a ligand, wherein the ligand is capable ofbinding to the mutant polypeptide, thereby stabilising the fusionpolypeptide. In the absence of ligand, the fusion polypeptide is notstabilised, hence the polypeptide of interest is not active. The ligandis preferably a DHFR inhibitor. The fusion polypeptides used in suchsystems may be as described in the section “Fusion polypeptides”.

Also disclosed herein is the use of such a system to conditionallystabilise a polypeptide of interest.

As can be seen in the examples (table 2), a fluorescent reporter proteinfused to a Y100E DHFR mutant polypeptide has a maximal activity in thepresence of ligand which is 8.93-fold higher than in the absence ofligand. The present DHFR mutant polypeptides may in some embodimentsresult in an increased ratio between the maximal activity of apolypeptide of interest in the presence of ligand and its maximalactivity in the absence of ligand compared to what is observed for apolypeptide of interest fused to a Y100E DHFR mutant polypeptide. Insome embodiments, the maximal activity of the polypeptide of interest inthe presence of ligand is at least 9-fold higher than in the absence ofligand, such as at least 10-fold, such as at least 11-fold, such as atleast 12-fold, such as at least 13-fold, such as at least 15-fold, suchas at least 16-fold, such as at least 17-fold, such as at least 18-fold,such as at least 19-fold, such as at least 20-fold, such as at least25-fold, such as at least 30-fold, such as at least 40-fold, such as atleast 50-fold. In some embodiments, the mutant polypeptide does notdiffer from SEQ ID NO: 1 and SEQ ID NO: 2 only by a Y100I mutation.

The activity of the fusion polypeptide, and hence of the polypeptide ofinterest, is dependent on the amount of ligand. The mutant polypeptidesdisclosed herein have a typical dose-response curve, representednormally by a sigmoidal curve, where the inflection point corresponds toEC₅₀. Typically, the response will saturate after a certain amount ofligand is added to the system, which corresponds to the maximal activitythe polypeptide of interest can attain in response to the ligand. Theresponses of the mutant polypeptides disclosed herein to a given ligandmay differ: the same ligand may have a greater potency for a givenmutant polypeptide than for another given mutant polypeptide, and viceversa. The greater the potency of a ligand is, the steeper the slope ofthe dose-response curve will be. The choice of mutant polypeptide maythus be guided by the type of response, e.g. the potency or amplitude,which it is desirable to achieve.

The activity (or stability) of the polypeptide of interest can bedetermined by methods known to the person of skill in the art. Suchmethods include enzymatic assays, spectrophotometric assays,chemiluminescent assays, calorimetric assays, binding assays, growthassays, differentiation assays. The activity of the polypeptide ofinterest may be determined indirectly by using a reporter polypeptide asdescribed herein, of which the activity/stability may be determined asknown in the art.

Preferably, the activity of the polypeptide of interest in the presenceof ligand is such that it can exert the desired effect, e.g. atherapeutic effect, when the ligand is administered to a subject in needof such an effect.

Ligand

The present systems are based on the interaction between the mutantpolypeptides and a DHFR ligand. Such a DHFR ligand may be an inhibitorof DHFR activity or it may be an enhancer of DHFR activity.

In particular embodiments, the DHFR ligand is a DHFR inhibitor selectedfrom the group consisting of trimethoprim (trade names Proloprim,Monotrim, Triprim), brodimoprim, tetroxoprim, iclaprim (codenamed AR-100and RO-48-2622), pyrimethamine (trade name Daraprim), proguanil (tradename Paludrine), methotrexate (formerly known as amethopterin, tradenames Trexall, Rheumatrex) and pemetrexed (trade name Alimta), oranalogues thereof.

The above inhibitors may be divided in two categories. Compounds of thefirst category are antibiotics and comprise trimethoprim (trade namesProloprim, Monotrim, Triprim), brodimoprim, tetroxoprim, iclaprim(codenamed AR-100 and RO-48-2622). These may be particularlyadvantageous because they are not expected to exert any activity on amammalian host cell, and are thus “biologically silent” whenadministered to a mammalian subject.

Compounds of the second category comprise pyrimethamine (trade nameDaraprim), proguanil (trade name Paludrine), methotrexate (formerlyknown as amethopterin, trade names Trexall, Rheumatrex) and pemetrexed(trade name Alimta). These are anti-cancer or anti-malarial drugs.

Accordingly, in some embodiments the system for conditionallystabilising the fusion polypeptide comprises a fusion polypeptide, apolynucleotide encoding a fusion polypeptide, a vector comprising such apolynucleotide or a gene expression system as described herein above,and a ligand capable of binding to the mutant polypeptide, wherein theligand is a DHFR inhibitor selected from the group consisting oftrimethoprim (trade names Proloprim, Monotrim, Triprim), brodimoprim,tetroxoprim, iclaprim (codenamed AR-100 and RO-48-2622), pyrimethamine(trade name Daraprim), proguanil (trade name Paludrine), methotrexate(formerly known as amethopterin, trade names Trexall, Rheumatrex) andpemetrexed (trade name Alimta). Preferably, the ligand of the system istrimethoprim, brodimoprim, tetroxoprim or iclaprim. In one embodimentthe system comprises trimethoprim as ligand.

Treatment of a Disorder

Mutant polypeptides, fusion polypeptides, systems for gene expression orsystems for conditionally stabilising a fusion polypeptide, and vectorscomprising a polynucleotide encoding the fusion polypeptides are alsoprovided for use in the treatment of a disease or disorder in a subjectin need thereof. A method of treatment of a disease or disorder in asubject in need thereof, comprising conditionally stabilising apolypeptide of interest using the systems described herein, is alsoprovided.

The disease or disorder may be associated with reduced or absentactivity of the polypeptide of interest, or of a protein comprising thepolypeptide of interest, in the subject in need of treatment. In caseswhere the polypeptide of interest has e.g. enzymatic activities, thedisorder or disease may be associated with the lack of or with reducedlevels of a product of a reaction catalysed by the polypeptide ofinterest.

The subject in need of treatment is any subject suffering from orsuspected of suffering from a disease or disorder, for which it may bedesirable to conditionally stabilise a polypeptide of interest asdescribed herein.

In some embodiments, the treatment comprises administering apolynucleotide encoding a fusion polypeptide as described herein or avector comprising such a polynucleotide in a subject. Such methods havebeen described above and are known the person of skill in the art andencompass ex vivo gene therapy, where the fusion polypeptide or vectoris administered indirectly by transplanting cells capable of expressingthe fusion polypeptide or comprising the vector. The treatment furthercomprises administering the ligand as described above, such as a DHFRinhibitor, e.g. trimotheprim or any DHFR inhibitor described above, tothe subject. The preferred mode or route of administration can bedetermined by the person skilled in the art and may vary depending onthe nature of the disorder or disease to be treated, on the subject, onthe vector used and on the ligand.

As explained herein above, the present systems are particularlyadvantageous in that several mutant polypeptides are provided, which maydisplay different responsiveness to a given ligand. The present systemscan thus be precisely tuned in order to match the optimal administrationdosage for a given subject, depending on the nature of the subject andhis/her health condition, the nature of the disorder, the nature of theligand, and other factors as evident to the skilled person.

Examples

Plasmids

Gene synthesis and site-directed mutagenesis (Genscript) were used togenerate E. coli DHFR (GenBank: J01609.1, nucleotides 558-1033) andvariants, which were inserted into a plasmid with two expressioncassettes. The cassettes consisted of the human CMV promoter (GenBank:KJ872540.1, nucleotides 175075-174488) driving mCherry (GenBank:HM771696.1, nucleotides 7106-7813) expression and the murine CMVpromoter (GenBank: U68299.1, nucleotides 183388-182860) drivingexpression of respective DHFR variants fused to the N-terminus of YFP(GenBank: KJ411637.1, nucleotides 5913-6632). A set of plasmids withDHFR variants were generated, which contained GCH1 (NCBI ReferenceSequence: NM_000161.2, nucleotides 162-913) instead of YFP, but wereotherwise the same. The following control plasmids were used: a plasmidwith only the mCherry expression cassette (pmC), a plasmid with only theYFP expression cassette (no DHFR) (pY), a plasmid with both the mCherryand YFP expression cassettes (no DHFR) (pmCY), a plasmid with themCherry expression cassette and the human CMV promoter driving TH (NCBIReference Sequence: NM_000360.3, nucleotides 20-1513) expression(pmC-TH) and a plasmid with the mCherry expression cassette and thehuman CMV promoter driving GCH1 expression (pmC-GCH). All expressioncassettes included WPRE (GenBank: J04514.1, nucleotides 1093-1684) and asynthetic polyA signal sequence (Proudfoot et al 1989). Fused genes werelinked by a 15 amino acid sequence coding for GGGGSGGGGSGGGGS.

Cell Culture

HEK 293 cells were grown as adherent culture using Nunclon flasks and24-well plates (Thermo Fisher Scientific). The medium consisted of highglucose Dulbecco's modified Eagle's medium with non-essential aminoacids, Phenol red, 2 mM L-glutamine and 5% fetal bovine serum (Gibco).Cells were cultured in a humidified incubator at 37° C. with 5% CO₂ andpassaged using 0.05% trypsin with 0.48 mM EDTA (Gibco).

Cells were transfected with plasmids for DHFR variant analysis in24-well plates using Lipofectamine 2000 (Invitrogen) according to themanufacturer's recommendations. 0.5 plasmid and 1.5 μl Lipofectamine2000 were used per well. TMP was dissolved in culture medium at aconcentration of 120 μM and were added to the cell culture to aconcentration of 10 μM, 24 hours after transfection. Cells wereharvested using trypsinization 2 days after transfections andre-suspended in DPBS with 2% fetal bovine serum and 1 mM EDTA, followedby 50 μm filtration. Draq7 (Abcam) was mixed into the cell solutions toa concentration of 0.3 μM just prior to flow cytometry analysis.

Flow Cytometry

YFP and mCherry expression of cells were quantified by flow cytometryusing a BD FACS Aria III and the BD FACSDiva 6.1.3 software. YFPfluorescence was detected using a 488 nm laser and a 530/30 nm filter.mCherry fluorescence was detected using a 633 nm laser and a 660/20 nmfilter. Draq7 was used to analyze viability and the fluorescence wasdetected using a 633 nm laser and a 730/45 nm filter. Color compensationwas applied for mCherry spillover into Draq7.

Sample Preparation for Combined HPLC-ECD and HPLC-MS Analysis of Mediaand Cells

Cell pellets were analyzed for BH4 content by first adding 10 μl 100 mMHCl+5 mM ascorbic acid+50 μl 5 mM HCl+5 mM ascorbic acid to each cellpellet followed by ten 3 sec sonication runs while the samples were kepton ice. After incubation on ice for an additional 5 mins, the pellet wasseparated from the supernatant by centrifugation for 10 min at 14,000rpm in 4° C. The supernatant was then transferred to 4 kDa cut offfilter and spin down and stored at −80° C. until MS analysis.

Cell culture media was analyzed for DOPA after adding 5 μl 100 mM HCl+5mM ascorbic acid to 400 μl and filtration using Amicon 3 kDa cut offspin columns after centrifugation for 1 h at 14,000 rpm in 4° C. Sampleswere stored at −80° C. until ECD analysis.

Variations to Residues in Loops

The following positions were identified as candidates for limited andrescuable destabilization in protein loops: R12, M42, E90, D127, W133,H141 and F153. These residues have varying numbers of interactions, suchas hydrogen bonds, with other residues. They are placed so that theystabilize the surrounding structures in loops or secondary structuralelements. There were two types of variations in these positions.Substitutions by alanine or glycine were made to remove the side chaininteractions and replacements by other residues were intended to weakenor reverse interactions in these positions.

We found that both M42G and M42A variants are destabilizing and can berescued by TMP. Variant M42A has higher ratio of rescue. The effect isincreased when combined with substitution Y100E, however the doublevariant with M42G has very low activity. When the side chain ofglutamate in position 90 (E90) is replaced by alanine the effect isstronger than that for aspartate. Aspartate has similar physicochemicalproperties as glutamate, however is shorter by one carbon atom andtherefore cannot form the same contacts as the end group of the longerside chain of glutamate. The side chain of E90 interacts with aminoacids from three different parts of the structure.

In the case of aspartate in position 127 (D127) the substitutions by Aand N were made. The former removes the side chain, whereas in D127N thecharge of carboxyl group is replaced by polar amino group. Both variantshad very similar profiles to that of the reference. Combination withY100E or Y100I did not have a major impact for the alanine variants;whereas Y100I together with the D127N had a significant destabilizingeffect that could be reversed by TMP binding.

TABLE 1 DFHR protein amino acid variation sites tested are listed below.In some of the positions more than one variation was tested and listedwith consecutive letters referring to each replacement for thatposition. Note also that some of the results presented here refer tovariants with more than one amino acid substitution. Amino acididentities follow international abbreviations standards and wild typesequence refers to UniProt entry DYR_ECOLI for Escherichia coli (strainK12) with accession number P0ABQ4. Structural Amino acid position andcontext variants tested Type of variant α-helix Y100 to I or E Cofactorbinding β-strand M42 to A or G Modification and stability of H141 to Gsurface loops F153 to A, D or G Loops R12 to Y or A Modification andstability of N18 to T surface loops E90 to A or D Structural changesD122 to A P126 to D, R or Y D127 to A or N W133 to A

Among the loop structure variations (Table 1), one of the most potentvariations is tryptophan to alanine substitution in position 133(W133A). W133 has extensive hydrogen bonding network formed both by thebackbone and side chain atoms. It is buried inside the folded structure.Thus substitution of W by A at this position removes the side chaininteractions. The stabilizing effect of TMP on this variant is thehighest (22.9 fold) among all the variants investigated here (FIG. 1).When W133A variant is combined with other substitutions, either thedestabilization is no longer reversible by binding of the inhibitor(Y100E, F153D, F153G) or the effect is significantly smaller (Y100I)(FIG. 2). Notably, removal of side chain interactions in position 141 bysubstitution of histidine with glycine (H141G) had an only modestdestabilizing effect that did not convey sufficient magnitude of controlon the TMP dependent stabilization of the protein (FIG. 1).

Phenylalanine in position 153 (F153) is located in the middle of theβ-strand and forms several aromatic-aromatic side chain interactionswith residues from two surrounding secondary structural elements.Substitutions of F153 by G, A or D were among the ones with strongesteffect (FIG. 1). Notably, when F153D and F153G were combined with eitherY100E or Y100I the constructs were no longer rescuable.

In conclusion, these results show that variations designed to affectresidues binding loop structures or secondary structural elements of theprotein core (namely the W133 and F153 residues) are thereforecandidates for generating destabilizing domains. Substitutions whichcompletely (by G) or almost completely (by A) remove the side chain andtherefore eliminate the side chain interactions with other amino acidsare among the best alterations. However, at some sites more subtlechanges (e.g., F153D) present properties fulfilling the main principlesof controlled stability, as well.

Combination with variants at the site detected in the previous patent(Y100) can rescue some variations that are not functional alone,however, they do not have positive impact on the strongest variantsdetected in here, in fact the addition of either Y100E or Y100I resultedin worse response characteristics for the respective the double variantand had reduced effect on rescue of stability by inhibitor binding (FIG.2).

An overview of the results is shown in Table 2.

TABLE 2 List of all single site variations and their effects on YFPfluorescence is shown in the presence and absence of TMP inhibitor usedas the stabilizing agent (left column). Variants that contain Y100Imodification (middle column) and those that contain Y100E variant (rightcolumn) are provided in this table. Mean of Mean of Mean of NormalizedY100I Normalized Normalized YFP ratio contain- YFP ratio Y100E YFP ratio(+) (−) ing (+) (−) containing (+) (−) variant TMP TMP variants TMP TMPvariants TMP TMP WT 1.513 1.434 R12Y 1.000 0.107 R12A 0.765 0.033 Y100IY100E (ref) R12A 1.384 1.115 R12A 1.068 0.996 N18T 0.782 0.119 Y100IY100E N18T 1.357 1.281 N18T 1.650 0.685 N18T 0.375 0.354 Y100I Y100ED122A M42A 1.493 1.120 N18T 1.282 0.821 M42A 0.678 0.039 Y100I Y100ED122A M42G 1.106 0.438 Y100I 1.428 1.023 M42G 0.049 0.027 D122A Y100EY100E 1.330 0.149 Y100I 1.447 1.153 Y100E 0.966 0.823 D127A D127A Y100I1.226 1.111 Y100I 0.809 0.090 Y100E 1.110 1.194 D127N D127N D122A 1.6121.487 Y100I 0.369 0.086 Y100E 0.257 0.241 W133A P126Y P126Y 1.499 0.583Y100I 0.086 0.081 Y100E 0.088 0.084 F153D P126D P126D 1.249 0.230 Y100I0.088 0.078 Y100E 0.273 0.201 F153G P126R P126R 1.430 0.383 Y100E 0.0760.091 W133A D127A 1.527 1.479 Y100E 0.056 0.059 F153D D127N 1.884 1.823Y100E 0.067 0.076 F153G W133A 1.196 0.052 F153G 1.089 0.053 F153A 1.1080.208 F153D 1.189 0.064

Variations to Inhibitor and Cofactor Binding Residues

We found that the variants modifying cofactor binding residues at N18 orD122 did not have wanted properties. Only the variants at position Y100could be used for stability changes as previously indicated. Both N18and D122 have contacts with other amino acids, however not as many asY100, which is at binding distance to numerous residues. Y100 interactsdirectly with NADPH and forms hydrogen bond with 15 and G96 and L104.

Replacement of N18 by threonine which is of same size but without thepolar amino groups has about the same properties as the reference.Together with Y100 variants the rescue effect is evident, especiallywith Y100E. Y100E variation alone is similar to the R12Y Y100Ireference, whereas Y100I alone showed no clear destabilization effect.

Next, the two Y100 variants were investigated together with severalother substitutions (see Table 2). The strongest destabilization effectappeared for the combined Y100E M42A substitution. However, in thiscase, the rescue by the inhibitor was compromised.

Variations at Structural Sites

Prolines are special amino acids due to forming ring with the proteinbackbone. They have limited flexibility and introduce special turns tostructures making the structure locally rigid. Proline 126 is in themiddle of extended loop structure. Variations Y, D and R all have usefulproperties. When proline is substituted in this position, alterations tothe backbone as well as to the interactions were expected. P126 hadinteraction also with Y100 and with numerous bonds with neighbouringF125. These interactions likely keep the protein tightly packed. Thesubstitutions by D, R and Y likely affect the intramolecular bonds andrender the molecule less stable. This effect can be reversed by bindingTMP to the protein. Introduction of Y100E variation together with thethree P126 substitutions had similar properties to the reference.

CONCLUSION—RATIONAL DESIGN OF STABILITY AFFECTING VARIATIONS

The goal of this study was to substitute amino acid residues in arational way, so that binding of the inhibitor to the structure wouldrestore the stability, structure and function. It is essential to keepthe binding site intact and therefore no major changes were made in thisarea. Instead, we concentrated on amino acids that have severalinteractions with different parts of the molecule thereby beingessential for stability. These sites locate mainly on loop structures,although some of them are in secondary structural elements (β-strands)and binding surface loops to the protein core. The idea in addressingthese sites is to make the structure locally more flexible and thus lessstable. Inhibitor binding would help in locking the structure to thestable normal conformation. In the design a delicate balance is neededto avoid destroying the structure to such an extent that it cannot berescued, while on the other hand significantly changing stability.

The design process was initiated by searching for amino acids outsidethe immediate cofactor and ligand/inhibitor binding site that haveinteractions, mainly hydrogen bonds but also salt bridges andaromatic-aromatic interactions, with other parts of the structure. Theseresidues are structurally important and therefore alterations at themwould reduce stability. We did not want to make major structural changesas they were considered to be too large to allow rescue. Residues withseveral side chain interactions with multiple parts of the molecule wereprioritized.

The replacements were designed to remove side chain interactions. Thisis achieved by removing the side chain and replacing by glycine oralanine which do not have any carbon groups or just one in the sidechain. These residues cannot form the interaction networks of the wildtype side chains of larger residues. At some places also other types ofsubstitutions were introduced, for example to replace the chargedcarboxyl groups of aspartate and glutamate by amino groups of asparagineor glutamine. These alterations would retain most of the side chaininteractions but some interactions would either be missing or be weaker.

In conclusion, the design principles were

-   -   to select residues having side chain interactions with other        parts of the protein    -   to avoid changes to the binding sites    -   to remove or shorten amino acid side chains so that interactions        cannot be formed    -   to replace interacting groups with others that could form weaker        interactions

The rational design approach requires knowledge about the proteinstructure including structures with binding molecules (ligands,inhibitors, cofactors) based on experimental structure(s) or highquality computational model(s).

SequencesSEQ ID NO: 1 (UniProt P0ABQ4) >sp|P0ABQ4|DYR_ECOLI Dihydrofolate reductaseOS = Escherichia coli (strain K12) GN = folA PE = 1 SV = 1MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHS YCFEILERRSEQ ID NO: 2 >ECOLI Dihydrofolate reductase R12Y G67S mutantMISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHS YCFEILERRSEQ ID NO: 3 >Exemplary linker GGGGSGGGGSGGGGS

REFERENCES

-   Bachmair, A. et al. (1986). Science 234:179-186.-   Dohmen, R. J. et al. (1994) Science 263:1273-1276-   Fire, A. et al. (1998) Nature 391:806-811. [0025]-   Janse, D. M. et al. (2004) J. Biol. Chem. 279:21415-21420.-   Kanemaki, M. et al. (2003) Nature 423:720-724.-   Labib, K. et al. (2000) Science 288:1643-1646.-   Medema, R. H. (2004) Biochem. J. 380:593-603.-   Park, E-C. et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:1249-1252.-   Ryding, A. D. S. et al. (2001) J. Endocrinol. 171:1-14.-   Schneekloth, J. S. et al. (2004) J. Am. Chem. Soc. 126:3748-3754.

1. A mutant polypeptide derived from DHFR comprising or consisting of asequence differing from SEQ ID NO: 1 and SEQ ID NO: 2 in at least one ofthe following positions: R12, N18, M42, Y100, D122, P126, D127, W133and/or F153, said sequence having at least 70% identity, such as atleast 75% identity, such as 80% identity, such as at least 85% identity,such as at least 90% identity, such as at least 95% identity, such as atleast 96% identity, such as at least 97% identity, such as at least 98%identity, such as at least 99% identity to SEQ ID NO: 1 or SEQ ID NO: 2,with the proviso that the mutant polypeptide does not differ from SEQ IDNO: 1 and SEQ ID NO: 2 only by a Y100I mutation.
 2. The polypeptide ofclaim 1, wherein the sequence of the polypeptide differs from SEQ ID NO:1 and SEQ ID NO: 2 in at least one of the following positions: W133 andF153
 3. The polypeptide of any one of the preceding claims, wherein themutant further comprises at least one mutation at position Y100, such asY100E or Y100I, preferably Y100E.
 4. The polypeptide of any one of thepreceding claims, wherein the sequence differs from SEQ ID NO: 1 by thepresence of a mutation which at least partly removes the side chain atthe mutated position.
 5. The polypeptide of any one of the precedingclaims, wherein the mutation which at least partly removes the sidechain is a mutation to a glycine G or to an alanine A in at least one ofpositions R12, N18, M42, P126, D127, W133 and/or F153.
 6. Thepolypeptide of any one of the preceding claims, wherein the mutantcomprises one or more of the mutations selected from the groupconsisting of: W133A, F153G, F153D, R12A, M42A, D122A, P126Y, P126D,P126R, F153A, M42A Y100E, R12A Y100E, N18T Y100I and D127N Y100I.
 7. Thepolypeptide of any one of the preceding claims, wherein the mutantfurther comprises a mutation in the TMP binding pocket of SEQ ID NO: 1.8. The polypeptide of any one of the preceding claims, wherein thepolypeptide does not have a G67S mutation.
 9. The polypeptide of any oneof claims 1 to 2, wherein the polypeptide does not have a Y100 mutation.10. The polypeptide of any one of the preceding claims, wherein thesequence differs from SEQ ID NO: 1 or SEQ ID NO: 2 at least in positions150 and W133, or at least in positions R52 and W133, or at least inpositions L54 and W133, or at least in positions P55 and W133,preferably the sequence differs from SEQ ID NO: 1 or SEQ ID NO: 2 inpositions 150 and W133, or in positions R52 and W133, or in positionsL54 and W133, or in positions P55 and W133.
 11. The polypeptide of anyone of the preceding claims, wherein the sequence differs from SEQ IDNO: 1 or SEQ ID NO: 2 at least in positions 150 and F153, or at least inpositions R52 and F153, or at least in positions L54 and F153, or atleast in positions P55 and F153, preferably the sequence differs fromSEQ ID NO: 1 or SEQ ID NO: 2 in positions 150 and F153, or in positionsR52 and F153, or in positions L54 and F153, or in positions P55 andF153.
 12. The polypeptide of any one of the preceding claims, whereinthe mutant differs from SEQ ID NO: 1 and SEQ ID NO: 2 in at least onefurther position, such as at least two further positions, such as atleast three further positions.
 13. The polypeptide of any one of thepreceding claims, wherein the further position is I50, R52, L54 or P55of SEQ ID NO: 1 or SEQ ID NO:
 2. 14. The polypeptide of any one of thepreceding claims, wherein the mutant polypeptide is a W133A mutant ofSEQ ID NO: 1, an F153G mutant of SEQ ID NO: 1, an F153D mutant of SEQ IDNO: 1, an R12A Y100E mutant of SEQ ID NO: 1, an N18T Y100E mutant of SEQID NO: 1, an M42A Y100E mutant of SEQ ID NO: 1, a Y100I D127N mutant ofSEQ ID NO: 1, or a Y100I W133A mutant of SEQ ID NO:
 1. 15. A fusionpolypeptide comprising the mutant polypeptide of any one of claims 1 to14 operatively linked to a polypeptide of interest.
 16. The fusionpolypeptide of claim 15, wherein the mutant polypeptide is furtherlinked to the N terminus of the polypeptide of interest.
 17. The fusionpolypeptide of any one of claims 15 to 16, wherein the mutantpolypeptide is further linked to the C terminus of a further polypeptideof interest.
 18. The fusion polypeptide of any one of claims 14 to 7,wherein the polypeptide of interest and/or the further polypeptide ofinterest is a polypeptide selected from the group consisting of: atherapeutic polypeptide, a reporter polypeptide, an enzyme and atranscription factor.
 19. The fusion polypeptide of any one of claims 14to 18, wherein the therapeutic polypeptide is a transcription factor, aneurotrophic factor, a cell surface receptor, an ATPase, acyclin-dependent kinase inhibitor or an antibody.
 20. The fusionpolypeptide of any one of claims 14 to 19, further comprising a signalpeptide capable of causing secretion of the fusion polypeptide from amammalian cell or a nuclear localisation signal capable of causingimport of the fusion polypeptide in the nucleus.
 21. The fusionpolypeptide of any one of claims 14 to 20, wherein the polypeptide ofinterest is additionally operatively linked to a reporter polypeptide.22. The fusion polypeptide of any one of claims 14 to 21, wherein thereporter polypeptide is a fluorescent protein, an enzyme or an affinitytag.
 23. A polynucleotide encoding the polypeptide of any one of claims1 to 14 or the fusion polypeptide of any one of claims 15 to
 22. 24. Agene expression system comprising a polynucleotide according to claim23.
 25. A system for conditionally stabilising a fusion polypeptidecomprising a polypeptide according to any one of claims 15 to 22 and aligand, wherein the ligand is capable of binding to the polypeptide andstabilising the fusion polypeptide.
 26. Use of the system according toclaim 25 to conditionally stabilise said polypeptide of interest. 27.The use of claim 26, wherein the maximal activity of the polypeptide ofinterest in the presence of ligand is at least 9-fold higher than in theabsence of ligand, such as at least 10-fold, such as at least 11-fold,such as at least 12-fold, such as at least 13-fold, such as at least15-fold, such as at least 16-fold, such as at least 17-fold, such as atleast 18-fold, such as at least 19-fold, such as at least 20-fold, suchas at least 25-fold, such as at least 30-fold, such as at least 40-fold,such as at least 50-fold.
 28. The use of any one of claims 26 to 27,wherein the maximal activity of the polypeptide is determined by anassay such as an enzymatic assay, a spectrophotometric assay, achemiluminescent assay, a calorimetric assay, a binding assay, a growthassay or a differentiation assay.
 29. The use of any one of claims 26 to28, wherein the fusion polypeptide further comprises a reporter gene andwherein the activity of the polypeptide of interest is determined bymeasuring the activity of said reporter gene.
 30. The use of any one ofclaims 26 to 29, wherein the ligand is a dihydrofolate reductaseinhibitor such as an antibiotic, preferably trimethoprim, brodimoprim,tetroxoprim, iclaprim, or pyrimethamine, or an anti-cancer drug, such asproguanil, methotrexate and pemetrexed, preferably trimethoprim.
 31. Theuse of claim 30 wherein the dihydrofolate reductase inhibitor is anantibiotic such as trimethoprim, brodimoprim, tetroxoprim, iclaprim orpyrimethamine, preferably trimethoprim.
 32. A vector comprising apolynucleotide encoding the fusion polypeptide of any one of claims 15to
 22. 33. The polypeptide of any one of claims 1 to 14, the fusionpolypeptide of any one of claims 15 to 22, the system of claim 25, orthe vector of claim 32, for use in the treatment of a disease in asubject in need thereof.
 34. The polypeptide, fusion polypeptide, systemor vector for the use of claim 33 wherein the disease is associated withreduced activity of the polypeptide of interest, or of a proteincomprising the polypeptide of interest.
 35. The polypeptide, fusionpolypeptide, system or vector for the use of any one of claims 33 to 34,wherein the treatment further comprises administering to the subject aligand capable of binding to and stabilising the fusion polypeptide. 36.An isolated host cell capable of expressing the fusion polypeptide ofany one of claims 15 to
 22. 37. A method of treatment of a disease in asubject in need thereof, comprising administration of polypeptide of anyone of claims 1 to 14, the fusion polypeptide of any one of claims 15 to22, the system of claim 25, or the vector of claim 32.